Huntington disease (HD) was the first disorder shown to be due to an expansion of a (CAG)n tract within exon 1 of the affected gene and to a resultant increase in length of a Qn domain within the corresponding encoded protein, huntingtin (Huntington""s Disease Collaborative Research Group, xe2x80x9cA Novel Gene Containing a Trinucleotide Repeat that is Expanded and Unstable on Huntington""s Disease Chromosome,xe2x80x9d Cell 72:971-983 (1993)). Subsequently, seven other neurodegenerative diseases, including dentatorubral pallidoluysian atrophy, spinobulbar muscular atrophy, and spinocerebellar ataxis 1, 2, 3, 6 and 7, have been shown to be caused by pathological (CAG)n/Qn-expansions (Lunkes et al., xe2x80x9cPolyglutamines, Nuclear Inclusions and Degenerations,xe2x80x9d Nat. Med. 3:1201-1202 (1997)).
Green was the first to suggest that transglutaminase(s) may play a role in trinucleotide-repeat diseases (Green, xe2x80x9cHuman Genetic Diseases Due to Codon Reiteration: Relationship to an Evolutionary Mechanism,xe2x80x9d Cell 74:955-956 (1993)). Others have also suggested that transglutaminase activity may be a factor in the etiology of (CAG)n/Qn-expansion diseases (Lorand, L., xe2x80x9cNeurodegenerative Diseases and Transglutaminase,xe2x80x9d Proc. Natl. Acad. Sci. USA 93:14310-14313 (1996); Cooper et al., xe2x80x9cPolyglutamine Domains are Substrates of Tissue Transglutaminase. Does Transglutaminase Play a Role in Expanded CAG/poly-Q Neurodegenerative Diseases?,xe2x80x9d J. Neurochem. 69:431-434 (1997)) and in the formation of proteinaceous deposits in the brains of Alzheimer disease patients (Selkoe et al., xe2x80x9cAlzheimer""s Diseases: Insolubility of Partially Purified Helical Filaments in Sodium Dodecyl Sulfate and Urea,xe2x80x9d Science 215:1243-1245 (1982) and Johnston et al., xe2x80x9cTransglutaminase Activity is Increased in Alzheimer""s Disease Brain,xe2x80x9d Brain Res. 751:323-329 (1997)). In support of the idea that transglutaminases may be involved in (CAG)n/Qn-expansion diseases, Qn domains, whether flanked by small numbers of amino acids (Kahlem et al., xe2x80x9cPeptides Containing Glutamine Repeats are Substrates for Transglutaminase-catalyzed Cross-linking: Relevance to Diseases of the Nervous System,xe2x80x9d Proc. Natl. Acad. Sci. USA 93:14580-14585 (1996) and Kahlem et al., xe2x80x9cTransglutaminase Action Imitates Huntington""s Disease: Selective Polymerization of Huntington Containing Polyglutamine,xe2x80x9d Mol. Cell 1:595-601 (1998)), or embedded within full-length protein constructs (Cooper et al., xe2x80x9cPolyglutamine Domains are Substrates of Tissue Transglutaminase. Does Transglutaminase Play a Role in Expanded CAG/poly-Q Neurodegenerative Diseases?,xe2x80x9d J. Neurochem. 69:431-434 (1997) and Cooper et al., xe2x80x9cTransglutaminase-catalyzed Inactivation of Glyceraldehyde 3-Phosphate Dehydrogenase Complex by Polyglutamine Domains of Pathological Length,xe2x80x9d Proc. Natl. Acad. Sci. USA 94:12604-12609 (1997)), are excellent substrates of tissue transglutaminase. It has been also shown that the ability of xe2x80x9cnakedxe2x80x9d Qn domains to act as Q-donor substrates of tissue transglutaminase increases in the order Q20 less than Q40 less than Q60 (Gentile et al., xe2x80x9cTissue Transglutaminase-catalyzed Formation of High-Molecular-Weight Aggregates In Vitro is Favored with Long Polyglutamine Domains: A Possible Mechanism Contributing to CAG-triplet Diseases,xe2x80x9d Arch. Biochem. Biophys. 352:314-321 (1998)). Moreover, the substrate activity per individual Q residue is greater for Q60 than for Q20 (Gentile et al., xe2x80x9cTissue Transglutaminase-catalyzed Formation of High-Molecular-Weight Aggregates In Vitro is Favored with Long Polyglutamine Domains: A Possible Mechanism Contributing to CAG-triplet Diseases,xe2x80x9d Arch. Biochem. Biophys. 352:314-321 (1998)). Importantly, Kahlem et al. showed that huntingtin containing a pathological-length Qn domain is a much better substrate of commercial guinea pig liver tissue transglutaminase and of crude brain tissue transglutaminase than is huntingtin containing a non-pathological-length Qn domain (Kahlem et al., xe2x80x9cTransglutaminase Action Imitates Huntington""s Disease: Selective Polymerization of Huntington Containing Polyglutamine,xe2x80x9d Mol. Cell 1:595-601 (1998)). Thus, pathological-length Qn domains may be synergistically better substrates of tissue transglutaminase than are non-pathological-length Qn domains.
Transglutaminase activity is known to be present in both the cytosolic and nuclear fractions of a variety of cells, including brain cells (Singh et al., xe2x80x9cIdentification and Characterization of an 80 Kilodalton GTP-binding Protein/Transglutaminase from Adult Rabbit Liver Nuclei,xe2x80x9d Biochemistry 34:15863-15871 (1995); and Lesort et al., xe2x80x9cDistinct Nuclear Localization and Activity of Transglutaminase,xe2x80x9d J. Biol. Chem. 273:11991-11994 (1998)). These are the same compartments in which aggregates are formed in affected brain cells.
A method for diagnosing HD or a predisposition to develop HD involves determining the number of (CAG)n repeats present in the huntingtin gene of a patient. This method is performed by using a fragment of a nucleic acid encoding hungtingtin in a gene amplification assay or other nucleic hybridization assays. This methodology is disclosed, for example, in U.S. Pat. Nos. 5,693,757 and 5,686,288 to MacDonald et al.
However, as noted above, there are a number of (CAG)n/Qn-expansion diseases, all of which involve different genes and proteins. Diagnostic procedures using nucleic acids for one (CAG)n/Qn-expansion disease would not necessarily identify all diseases. Rather than rely on a battery of nucleic acid-based procedures to diagnose a (CAG)n/Qn-expansion disease, a single diagnostic analysis would be beneficial. In particular, it would be beneficial to obtain a quick and reliable method of diagnosing (CAG)n/Qn expansion disorders, including disease stages, and assessing the efficacy of therapeutic treatments of the (CAG)n/Qn expansion disorders. The present invention overcomes these and other deficiencies in the prior art.
One aspect of the present invention relates to a method of diagnosing a (CAG)n/Qn-expansion disorder in an individual. This method is performed by obtaining a sample from an individual and determining whether the sample contains a concentration of Nxcex5-(L-xcex3-glutamyl)-L-lysine which is elevated in comparison to a normal concentration of Nxcex5-(L-xcex3-glutamyl)-L-lysine.
Another aspect of the present invention relates to a method of measuring progression of a (CAG)n/Qn-expansion disorder (i.e., for individuals previously diagnosed with a (CAG)n/Qn-expansion disorder). This method is performed by obtaining a first sample from an individual, determining a first concentration, of Nxcex5-(L-xcex3-glutamyl)-L-lysine in the first sample, obtaining a second sample from the individual at a time after the first sample is obtained, and determining a second concentration of Nxcex5-(L-xcex3-glutamyl)-L-lysine in the second sample. Progression of the (CAG)n/Qn-expansion disorder is indicated by the second concentration being higher than the first concentration.
Still another aspect of the present invention relates to a method of measuring therapeutic efficacy of a treatment for a (CAG)n/Qn-expansion disorder. This method is performed by obtaining a first sample from an individual, determining a first concentration of Nxcex5-(L-xcex3-glutamyl)-L-lysine in the first sample, providing the individual with a treatment for a (CAG)n/Qn-expansion disorder, obtaining a second sample from the individual after providing the individual with the treatment, determining a second concentration of Nxcex5-(L-xcex3-glutamyl)-L-lysine in the second sample, and comparing the second concentration to the first concentration to measure the therapeutic efficacy of the treatment for a (CAG)n/Qn-expansion disorder.
As indicated previously, there are currently a number of diseases or disorders that have been classified as (CAG)n/Qn-expansion disorders, particularly neurodegenerative diseases. Each year, numerous patients are diagnosed with these and other various neurodegenerative diseases. The present invention provides a reliable method of diagnosing whether an individual suffers from a (CAG)n/Qn-expansion disorder, as well as the progression thereof, and any treatments thereof.